Alzheimer’s disease is defined by the pathological aggregation of amyloid-beta and hyperphosphorylated tau. AD patients often exhibit other symptoms like metabolic and sleep dysfunction. Currently, it is unclear if impairments are a cause or consequence of Aβ or tau aggregation. Previously, we demonstrated cerebral and peripheral changes in glucose elevate brain lactate levels stimulating wake. Further, previous work showed tau pathology corresponds with decreased sleep. Currently, it’s unclear what mechanisms drive sleep changes or if it relates to metabolic dysfunction. Therefore, we investigated how tau pathology impacts sleep and metabolism in P301S mice. To examine peripheral metabolism, glucose tolerance tests were conducted on P301S and wild-type mice(female). Recordings of indirect calorimetry over 3 days further explored peripheral metabolism. Paired glucose and lactate biosensors in the hippocampi tracked sub-second fluctuations of brain interstitial fluid (ISF) to observe cerebral metabolic changes. EEG and EMG recorded cortical activity over a 3-day period and spectral analysis investigated sleep/wake architecture. Tau aggregation preserves peripheral glucose tolerance and diurnal rhythms of brain glucose/lactate, normally lost in aged controls, suggesting increased glucose utilization. Similarly, P301S mice show increased respiratory exchange ratio suggesting heightened carbohydrate utilization. Conversely, they exhibit diminished energy expenditure. EEG spectral analysis revealed increased delta, increased theta, and decreased beta with pathology, suggesting disrupted GABA activity and sleep drive. Starting early in pathology, mice display increased wake and decreased NREM/REM. Effects on total sleep depend heavily on decreased REM bout number and duration. Blood glucose levels in P301S mice fail to rise in response to glucose injection. Brain glucose/lactate fluctuations are maintained suggesting tau pathology preserves glucose utilization. Conversely, decreased NREM and REM and increased wake were exhibited with tau pathology. EEG spectral analysis indicates decreased beta activity, suggesting diminished GABA activity. Increased theta during NREM suggests increased pressure to transition to REM sleep. Reduced sleep drive is illustrated by increased delta in wake. Together, tau pathology reduces sleep drive and ability to switch between vigilant states despite increased sleep need. These results suggest that tau pathology causes excitatory/inhibitory imbalance and sleep impairment but does not contribute to profound effects on cerebral metabolic rhythms.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A deposition. Genotype, age, and Aβ deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

Single cell RNA sequencing (scRNA-Seq) is useful to classify cell-specific gene expression profiles in kidney tissue. As viable cells are required, we report an optimized cell dissociation methodology and the necessity of screening tissue histology prior to scRNA-Seq. We demonstrate that glomerular injury can selectively reduce the appearance of groups of cells during analysis of cell clustering and we confirmed reductions in cell-specific markers among injured cells on kidney sections with fluorescence microscopy. Interpretation of scRNA-Seq results may be refined based upon these considerations.

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-β (Aβ) release, offering a mechanistic link between type 2 diabetes and Alzheimer’s disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2–/– mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in patients with diabetes or prediabetes.

Two biomarkers for Alzheimer’s disease (AD) are amyloid-beta (Aβ) and tau aggregation. AD patients often experience comorbidities like metabolic dysfunction and/or sleep-wake cycle disruption, but it is unclear whether this can be directly linked to Aβ or tau aggregation. Previous research shows mouse models overexpressing Aβ also experience metabolic and sleep dysfunction, independent of comorbidities like type-2-diabetes. Our lab previously demonstrated that central and peripheral metabolic changes are linked. This bidirectional relationship is well described in mouse models of Aβ overexpression; however, this relationship is relatively unexplored in mouse models of tauopathy. Therefore, our lab is investigating how central and peripheral metabolism relate to sleep-wake disruption in the P301S PS19 mouse. To investigate changes in peripheral metabolism, body weights are measured, and glucose tolerance tests are conducted on P301S PS19 and wild type female mice. Blood glucose levels are taken after fasting for four hours. Next, the mice are given an ip dose of 2g/kg glucose followed by blood glucose measurements in 15-minute increments for two hours. Paired glucose and lactate biosensors placed within the mouse hippocampus track second by second metabolic fluctuations over three circadian days. Along with biosensors, EEG and EMG electrodes are positioned to record sleep-wake cycles during this period. All methods completed on 3-, 6-, and 9-month-old P301S and wild type female mice. While current experiments are ongoing, P301S mice become glucose sensitive where WT mice become glucose intolerant with age. Early analysis suggests pathology and age-related differences in metabolism and sleep also develop over the circadian day. Our data suggests that tau aggregation increases peripheral glucose metabolism, in a manner different than Aβ pathology. Tau pathology disrupts normal sleep patterns where it decreases time spent in NREM and REM and alters EEG frequencies like delta, beta, and gamma. As age and pathology progress, cerebral metabolism of glucose and lactate is increased compared to age-matched WT mice. We hypothesize tau pathology induced hyperexcitability alters sleep/wake cycles, peripheral and cerebral metabolism, and exacerbates pathology. Our current research is valuable in expanding knowledge of the relationship between metabolism, sleep, and tau in Alzheimer’s disease.

ATP sensitive potassium (KATP) channels act as metabolic sensors to regulate cellular excitability. We recently demonstrated that neuronal KATP channels are composed of Kir6.2 subunits, are highly expressed on excitatory and inhibitory neurons, and are differentially expressed in the Alzheimer’s brain (Grizzanti et al. 2022). Mechanistically, we demonstrated that KATP channels couple changes in cerebral metabolism with neuronal activity and amyloid-beta (Aβ) release, suggesting a role for KATP channels in Alzheimer’s disease. Here, we extend these studies to explore how KATP channels contribute to excitatory/inhibitory (E/I) balance in the brain to impact sleep and Alzheimer’s pathology. How KATP channels coordinate sleep, metabolism, and neuronal activity was examined using the Kir6.2-/- mice, a mouse model lacking neuronal KATP channel activity. Intracranial biosensors measuring interstitial fluid (ISF) glucose and ISF lactate concurrent with EEG/EMGs were implanted into the hippocampus of wild type (WT) and Kir6.2-/- mice. Diurnal rhythms of ISF glucose, ISF lactate, and EEG/EMG were recorded for 72 hours. Mice were injected with saline, glucose, or glibenclamide to determine effects of metabolic challenges on ISF glucose, ISF lactate, and sleep/wake cycles. Quantitative EEG analysis and sleep staging was performed and correlated with ISF glucose and lactate over the 24 hour light/dark period. Wildtype mice have diurnal fluctuations in ISF glucose and lactate, with increases during the dark cycle when mice are awake and decreases in the light cycle when mice are asleep. In Kir6.2-/- mice lacking neuronal KATP channel activity, diurnal fluctuations in ISF glucose and lactate are lost. Kir6.2-/- mice spent more time in NREM sleep and less time awake than WT mice. Furthermore, reductions in absolute EEG power suggest changes in the brain’s E/I balance. Lastly, Kir6.2-/- mice were unresponsive to metabolic challenges, demonstrating that ISF lactate, a metabolic trigger for wakefulness, is controlled by KATP channels. We describe the role of neuronal KATP channels as metabolic sensors in coordinating sleep/wake architecture in mice. Ongoing studies are exploring the impact of KATP channels on sleep and brain excitability in AD.

ABSTRACT Increased neuronal excitability contributes to amyloid-β (Aβ) production and aggregation in the Alzheimer’s disease (AD) brain. Previous work from our lab demonstrated that hyperglycemia, or elevated blood glucose levels, increased brain excitability and Aβ release potentially through inward rectifying, ATP-sensitive potassium (K ATP ) channels. K ATP channels are present on several different cell types and help to maintain excitatory thresholds throughout the brain. K ATP channels are sensitive to changes in the metabolic environment, which are coupled to changes in cellular excitability. Therefore, we hypothesized that neuronal K ATP channels are necessary for the hyperglycemic-dependent increases in extracellular Aβ and eliminating K ATP channel activity will uncouple the relationship between metabolism, excitability, and Aβ pathology. First, we demonstrate that Kir6.2/ KCNJ11 , the pore forming subunits, and SUR1/ ABCC8 , the sulfonylurea receptors, are predominantly expressed on excitatory and inhibitory neurons in the human brain and that cortical expression of KCNJ11 and ABCC8 change with AD pathology in humans and rodent models. Next, we crossed APP/PS1 mice with Kir6.2 -/- mice, which lack neuronal K ATP channel activity, to define the relationship between K ATP channels, Aβ, and hyperglycemia. Using in vivo microdialysis and hyperglycemic clamps, we explored how acute elevations in peripheral blood glucose levels impacted hippocampal interstitial fluid (ISF) glucose, lactate, and Aβ levels in APP/PS1 mice with or without K ATP channels. Kir6.2+/+, APP/PS1 mice and Kir6.2-/-, APP/PS1 mice were exposed to a high sucrose diet for 6 months to determine the effects of chronic hyperglycemia on Aβ deposition. We found that elevations in blood glucose levels correlate with increased ISF Aβ, amyloidogenic processing of amyloid precursor protein (APP), and amyloid plaque pathology in APP/PS mice with intact K ATP channels. However, neither acute hyperglycemia nor chronic sucrose overconsumption raised ISF Aβ or increased Aβ plaque burden in APP/PS1 mice lacking Kir6.2-K ATP channel activity. Mechanistic studies demonstrate ISF glucose not only correlates with ISF Aβ but also ISF lactate. Without K ATP channel activity, ISF lactate does not increase during hyperglycemia, which correlates with decreased monocarboxylate transporter 4 (MCT4) expression, a lactate transporter responsible for astrocytic lactate release. This suggests that K ATP channel activity regulates ISF lactate during hyperglycemia, which is important for Aβ release and aggregation. These studies identify a new role for Kir6.2-K ATP channels in Alzheimer’s disease pathology and suggest that pharmacological antagonism of Kir6.2-K ATP channels holds therapeutic promise in reducing Aβ pathology, especially in diabetic and prediabetic patients.

ABSTRACT Hyperexcitability is a defining feature of Alzheimer’s disease (AD), where aberrant neuronal activity is both a cause and consequence of AD. Therefore, identifying novel targets that modulate cellular excitability is an important strategy for treating AD. ATP-sensitive potassium (K ATP ) channels are metabolic sensors that modulate cellular excitability. Sulfonylureas are K ATP channel antagonists traditionally used to combat hyperglycemia in diabetic patients by inhibiting pancreatic K ATP channels, thereby stimulating insulin release. However, K ATP channels are not limited to the pancreas and systemic modulation of K ATP channels has pleotropic physiological effects, including profound effects on vascular function. Here, we demonstrate that human AD patients have higher cortical expression of vascular K ATP channels, important modulators of vasoreactivity. We demonstrate that peripheral treatment with the sulfonylurea and K ATP channel inhibitor, glyburide, reduced the aggregation and activity-dependent production of amyloid-beta (Aβ), a hallmark of AD, in mice. Since glyburide does not readily cross the blood brain barrier, our data suggests that glyburide targets vascular K ATP channel activity to reduce arterial stiffness, improve vasoreactivity, and normalize pericyte-endothelial cell morphology, offering a novel therapeutic target for AD. Graphical abstract Targeting vascular K ATP channel activity for the treatment of Alzheimer’s disease pathology.

INTRODUCTION:APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy. METHODS:A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed. RESULTS:APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2. CONCLUSION:Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.

Background: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction. Methods: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction. Results: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction. Conclusion: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.

Background Kidney risk variants (KRVs) in the APOL1 gene are associated with mitochondrial dysfunction. However, the molecular spectrum of metabolites affected by the G1 and G2 KRVs, and the downstream mitochondrial pathways they affect, remain unknown. Methods We performed a metabolomics analysis using HEK293 Tet-on cells conditionally expressing APOL1 G0, G1, and G2 KRVs to determine the patterns of metabolites and pathways potentially involved in nephropathy. The Welch two-sample t test, matched-pairs t test, and two-way repeated measures ANOVA were used to identify differential metabolites. Random forest, a supervised classification algorithm that uses an ensemble of decision trees, and the mean-decrease-accuracy metric were applied to prioritize top metabolites. Results Alterations in the tricarboxylic acid cycle, increased fatty acid oxidation, and compromised redox homeostasis were the major pathways affected by overexpression of APOL1 KRVs. Conclusions Impairment of mitochondrial membrane respiratory chain complex I appeared to account for critical metabolic consequences of APOL1 KRVs. This finding supports depletion of the mitochondrial membrane potential, as has been reported.

African Americans frequently carry G1 and G2 renal‐risk variants of the apolipoprotein L1 (APOL1) gene. Two copies of G1 and/or G2 confer 5–29 fold higher risk of chronic kidney disease (CKD); however, only 20% of those carrying risk‐genotype develop CKD. This indicates that an additional stressor may facilitate development of CKD in those carrying G1/G2 risk variants. Considering that a) G1/G2 variants elicit mitochondrial dysfunction in renal cells, b) APOL1 function is pH sensitive and c) renal interstitial pH is <7, we hypothesized that acidic interstitial environment in the kidney may amplify mitochondrial dysfunction induced by G1/G2 variants. To test the hypothesis, we incubated human embryonic kidney (HEK293) cells expressing APOL1 in pH=6.8 or 7.4; we estimated early mitochondrial dysfunction by measuring mitochondrial network morphology with confocal microscopy (Fig.1). HEK293 cells stably expressing APOL1 G0, G1, G2 or empty vector (EV) under doxycycline (Dox) control were exposed to Dox for 4, 6 or 8hr, while cultured in media pH=6.8 or pH=7.4. Confocal images were acquired at 1000 magnification in live cells kept in an environmentally‐controlled chamber at each time point; FIJI software was used to quantify relative mitochondrial length (ratio of “rods” vs. “network‐branches”). We also ascertained that a) APOL1 expression was comparable regardless of pH or APOL1 genotype for up to 8hr post Dox‐induction using real‐time PCR and b) cell viability was comparable under all conditions using lactate‐dehydrogenase‐based cytotoxicity assay. Without Dox, relative mitochondrial length was similar regardless of APOL1 genotype or pH. However, after 6hr of Dox‐induction in media pH=6.8, G2‐expressing cells had shorter mitochondrial length (6.54±0.40) vs. EV (7.65±0.72, p=0.02) or G0 cells (7.46±0.31, p=0.003), indicating early mitochondrial fragmentation. After 8hr of doxycycline‐induction in media pH=6.8, mitochondrial length in G1 (6.21±0.26) and G2 cells (6.46±0.34) was lower than EV (7.13±0.32, p=0.002 and p=0.008, respectively) and G0 cells (7.22±0.45, p=0.003 and p=0.01, respectively; Fig. 2). At media pH 7.4, mitochondrial length was comparable up to 8hr of Dox‐induction, regardless of APOL1 genotype. We concluded that acidic pH may facilitate early mitochondrial dysfunction induced by G1/G2 APOL1 risk‐variants in renal cells. We propose that an acidic interstitial pH may facilitate adverse effects of APOL1 renal‐risk variants in the kidney, but not other organs expressing APOL1.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Viral infections can trigger chronic kidney disease (CKD) and the urine virome may inform risk. The Natural History of APOL1-Associated Nephropathy Study (NHAANS) reported that urine JC polyomavirus (JCPyV) associated with a lower risk of APOL1-associated nephropathy in African Americans. Herein, association was assessed between urine JCPyV with CKD in African Americans independent from the APOL1 genotype. Methods Quantitative polymerase chain reaction was performed for urinary detection of JCPyV and BK polyoma virus (BKPyV) in 200 newly recruited nondiabetic African Americans. A combined analysis was performed in these individuals plus 300 NHAANS participants. Results In the 200 new participants, urine JCPyV was present in 8.8% of CKD cases and 45.8% of nonnephropathy controls (P = 3.0 × 10-8). In those with APOL1 renal-risk genotypes, JCPyV was detected in 5.1% of cases and 40.0% of controls (P = 0.0002). In those lacking APOL1 renal-risk genotypes, JCPyV was detected in 12.2% of cases and 48.8% of controls (P = 8.5 × 10-5). BKPyV was detected in 1.3% of cases and 0.8% of controls (P  =  0.77). In a combined analysis with 300 NHAANS participants (n = 500), individuals with urine JCPyV had a 63% lower risk of CKD compared with those without urine JCPyV (odds ratio 0.37; P = 4.6 × 10-6). RNA fluorescence in situ hybridization confirmed the presence of JCPyV genomic DNA and JCPyV messenger RNA (mRNA) in nondiseased kidney. Conclusions Inverse relationships exist between JCPyV viruria and non-diabetic CKD. Future studies should determine whether renal inflammation associated with CKD is less permissive for JCPyV reactivation/replication or whether JCPyV is a marker of reduced host immune responsiveness that diminishes immune pathologic contributions to CKD.

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002–0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Objective— Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Approach and Results— Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca 2+ ] i ) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca 2+ ] i . Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. Conclusions— Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca 2+ ] i as well as by phosphoinositide-3 kinase-protein kinase B–induced eNOS phosphorylation. Both mitochondrial reactive oxygen species–dependent and –independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m2 (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m2 (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m2; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m2 for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.